VACCINE COMPOSITIONS COMPRISING LIVE BACTERIAL VECTORS FOR PROTECTION AGAINST YERSINIA PESTIS INJECTION

Abstract: Novel DNA constructs are provided that are capable of transforming microorganisms such that they can be used as live or attenuated vaccines which induce such immune response at mucosal surfaces. Further provided are such transformed microorganisms per se and vaccine compositions containing them. Preferred constructs of the invention are capable of transforming microorganisms such that they express F1 based protein while retaining a capability to establish themselves in human or animal gut environment. Several constructs have been identified that are capable of transforming gut dwelling organisms such as <u>S. typhimurium</u> or <u>S. typhi</u> to enable F1 antigen production, but most of these affect the organism such that it can no longer function effectively in the gut, at least in so far as it cannot express the antigen e.g. being unstable and losing plasmid.

Description: VACCINE COMPOSITIONS COMPRISING LIVE BACTERIAL VECTORS FOR PROTECTION AGAIN YERSINIA PESTIS INJECTION. The present invention relates to novel vaccines for provision of protection against infection with the organism Yersinia pestis (Y-_ pestis) and to compositions containing them. Particularly provided are parentally and orally active vaccines capable of offering protection against bubonic and pneumonic plague, particularly by induction of mucosal immunity in both humans and other animals. Y. pestis is the highly virulent causative organism of plague in a wide range of animals, including man. Infection with this organism results in a high rate of mortality. Studies have shown that the high virulence is due to a complex array of factors encoded by both the chromosome and three plasmids, including the Lcr genes (see Straley, (1991) Microb. Pathogen 10: ppδ7-91), a fibrinolysin (Sodeinde & Goguen, (1988) Infec. Immun 56: pp2743-2748) , and a capsule. The capsule surrounding Y. pestis cells is composed of a protein -polysaccharide complex, the protein component of which is known as Fraction 1 (Fl) (see Baker et al (1952) J. Immunol 68: ppl31-l45) which is only fully expressed at 37°C. This complex confers resistance to phagocytosis, possibly by forming aqueous pores in the membranes of phagocytic cells (Rodrigues et al (1992) J. Med. Biol. Res. 25: PP75~79) • Detection of antibodies to Fl is the basis of standard serological tests for the surveillance and diagnosis of plague as infected animals produce a strong humoral response to the antigen (Shepherd et al (1986) J. Clin. Microbiol. 24: PP1075-1078) ; Williams et al, (1982) Bull. World Health Organ. 64: PP745-752) . The current whole cell vaccines available for prevention of plague are highly heterogeneous, resulting in side effects which make them unsuitable for widespread use (Reisman (1970) J. Allergy 46: PP49-55); Meyer et al (1974) J. Infect. Dis. 129: SI3-SI8) ; Marshall et al (1974) J. Infect. Dis. 129: S19-S25). Fl has been proposed as being the primary immunogen in whole cell vaccines (Williams et al (1980) Bull. World. Health Organ. 58: PP753"756; Chen et al, (1976) J. Infect. Dis. 133: pp302-309) and may therefore be a suitable candidate on which to base an improved vaccine against plague. The cafl gene encoding Fl antigen has been cloned and sequenced (Galyov et al (1990) FEBS Letters 277: pp230-232) and recombinant Fl expressed and purified from E.coli induced a protective response in BALB/c mice sufficient to protect against challenge with 10 5 virulent plague bacilli (Simpson et al (1990) Am. J. Trop. Med. Hyg. 43(4): PP389-396) . Such resistance to infection by Y. pestis is correlated with high titres of Fl antibody. It is known to orally administer genetically engineered organisms which express antigenic proteins for the purpose of inducing antigen production (EP 0474891) and it is further known that use of such route may result in mucosal immunity (Cardenas &. Clements (1992) Clin. Microbiol Rev 5 (3) PP328-342 and (1992) Vaccine 10 (4) pp263) . Furthermore, it has been reported that attenuated bacteria have been prepared that are protective against enteric diseases, including genus Yersinia (US 6865709) . The current vaccine for plague is the Cutter vaccine which comprises formaldehyde killed plague bacilli and is administered to the body by intramuscular injection. However, parenteral immunisation, although effective in inducing systemic immunity, does not effectively induce mucosal immunity (McGhee et al, (1992) Vaccine 10., 75-88). So far no Y. pestis vaccine capable of producing a protective immune response at mucosal surfaces has been developed. The present inventors have now provided recombinant DNA constructs that when incorporated into DNA of a microorganism, particularly of a human or animal gut colonising microorganism, are capable of transforming it such that it is able to express a protein which produces a protective immune response against Yersinia pestis in the human or animal body when the gut colonising microorganism is administered by oral routes. Preferred forms of the present invention provide such DNA constructs that transform such a microorganism while allowing it to maintain its ability to colonise the human or animal gut and thus provide sustained administration, ie. by exposure of antigen to the human or animal body immune system, preferably with systemic invasion of the human or animal body. Further provided are vectors eg.plasmids, containing the DNA constructs of the invention, that are capable of transforming a human or animal gut colonising microorganism such that it is capable of expressing a protein which produces a protective immune response against Yersinia pestis in a human or animal body when the microorganism is administered by oral or parenteral routes, preferably allowing the microorganism to maintain ability to colonise the human or animal gut, and preferably subsequently systemically to invade the body. Still further provided are microorganisms, preferably human or animal gut colonising microorganisms, transformed with a vector containing recombinant DNA, eg. a plasmid containing recombinant DNA, according to the invention such that it is enabled to express a protein which produces a protective immune response against Yersinia pestis in a human or animal body when the microorganism is administered by oral or parenteral routes, and preferably allows the microorganism to maintain its ability to colonise the human or animal gut, and preferably invade systemically. The protective response provided preferably includes protection at mucosal surfaces. One preferred recombinant DNA, and plasmids comprising it, are characterised in that they comprise a lacZ promoter in frame with a sequence encoding for all or part of the cafl antigen. Still further preferred recombinant DNA utilises a caflR positive regulator derived from the Fl operon itself. It is further preferred that the caflM fragment is included for the purposes of assisting export of mature protein through the cell wall of the host organism, ie. the transformed cell and/or the caflA fragment that encodes proteins thought to be important in anchoring the Fl subunit to the cell wall. Where the recombinant DNA includes the lacZ promoter it is preferably incorporated into a vector such as pUClδ that has the all or part of the cafl encoding sequence inserted in frame with the lacZ promoter Obviously the microorganisms of the invention are preferably attenuated microorganisms not capable of causing disease in humans or animals, eg. such as the Salmonella aro A or aro C mutants, preferably of species Salmonella fyphimurium or a Salmonella tvphi. Preferred vaccine compositions are provided as such microorganisms together with a pharmaceutically acceptable carrier, eg. saline or buffer. Attenuated microorganisms such as S. tvphimurium have been well characterised as carriers for various heterologous antigens (Curtiss, (1990 ); New Generation Vaccines, Woodrow & Levine (eds) Marcel Dekker Inc. New York; Cardenas and Clements, (1992) ibid). Attenuation may be effected in a number of ways, such as by use of the aro A and/or aro C mutation approach (see Hosieth et al (1981) Nature 291, 238-239; Dougan et al (1986) Parasite Immunol 9, I51-I6O; Chatfield et al (1989) Vaccine 7, 495-498). Many other such attenuating deletions and mutations will be known for these and other microorganisms which will render them suitable for transformation with constructs of the present invention for the purposes of expressing vaccine proteins in the gut and/or gut colonisation in animals to be treated for Y. estis. with systemic invasion and colonisation following. For human vaccination attenuated S. tvohi is the preferred microorganism. A particularly preferred recombinant DNA, and plasmid or human or animal gut colonising microorganism incorporating it, encodes for or expresses all or part of the mature cafl (Fl) protein of Yersinia pestis. A particularly preferred recombinant DNA comprises a DNA sequence as described in SEQ ID No 4. The inventors have determined the sequence of a still further preferred recombinant DNA which when included within suitable vectors within, or integrated directly into the chromosomal DNA of, gut dwelling microorganisms results in still stronger expression of protective Fl, Fl fusion or Fl truncate proteins. This recombinant DNA is particularly characterised in that it it comprises the complete Fl operon including caflR, a positive regulator of Fl expression; caflM, encoding for the proposed chaperone sequence which assists in export of the Fl sub-units across the cell wall; caflA, thought to encode a protein which anchors the Fl into the cell wall; all in addition to the cafl gene encoding the Fl subunit or a truncate or fusion product thereof. The method, constructs, microorganisms and vaccines of the invention will now be exemplified by way of illustration only by reference to the following Sequence listing. Figure and Examples. Still further embodiments will be evident to those skilled in the art in the light of these. SEQUENCE LISTING: SEQ ID No 1: is the sequence of a PCR primer oligonucleotide corresponding to the first 21 bases encoding for mature cafl with an additional 5' region encoding for a Sacl site. SEQ ID No 2: is the sequence of a PCR primer oligonucleotide corresponding to the sequence of cafl which encodes a 'stem loop' downstream of the termination codon with an added 5' region encoding Sacl and Accl sites. SEQ ID No 3: is that of a PCR primer oligonucleotide corresponding to an internal end region of the cafl gene starting 107 bases downstream from the end of the first oligonucleotide. SEQ ID No 4: is that of the pFGAL2a construct showing the fusion of the first few bases of the β-galactosidase sequence in the vector with cafl minus its signal sequence and having a 5' tail including a Sac I restriction site; the sequence is shown up to the cafl AACC 3' end with some vector bases. SEQ ID No 5 is that of the protein encoded by pFGAL2a. SEQ ID No 6: is that of pFSIG3a: including cafl sequence encoding mature Fl expressed as a fusion with the E. coli LTB signal sequence encoded by the vector and having 5' tail including a Sacl restriction site; the sequence shown to AACC at 3' end of cafl and its adjoining vector bases. SEQ ID No 7: is that of the protein encoded by pFSIG3a. SEQ ID No 8: is that of pFORFlb: including the entire cafl gene and having a 5' tail including a Sacl restriction site; the sequence shown to TATAG downstream of the cafl open reading frame. The two series encoded at the 5' end of the sequence are produced separately to the Fl fusion. SEQ ID No 9 is that of the end of the first protein encoded by pFORFlb. SEQ ID No 10: is that of the Fl fusion encoded by pFORFlb. SEQ ID No 11: is that of primer FI0U2 used to amplify the Fl operon. SEQ ID No 12: is that of primer M4D used to amplify the Fl operon. SEQ ID No 13: is that of primer M3U used to amplify the Fl operon. SEQ ID No 14: is that of primer FI0D2 used to amplify the Fl operon. SEQ ID No 15: is that of a primer used with the primer of SEQ ID No 1 for preparation of pFSIG3a. SEQ ID No 16: is that of a primer used with the primer of SEQ ID No 17 to produce pFORFlb. SEQ ID No 17: is that of a primer used with the primer of SEQ ID No 16 to produce pFORFlb. FIGURE: Figure 1 shows schematic representations of the positions of Fl sequences in the constructs pFGAL2a, pFSIG3a and pFORFlb. Figure 2 shows to relative positions of the primers SEQ ID No 11 to 14, the retriction enzyme sites and the caflR, caflM, caflA and cafl subunits. EXAMPLES. General methods: Y. pestis was grown aerobically at 28°C in Blood Agar Base broth, pH6.8, containing 15g/l proteose peptone, 2.5g/l liver digest, 5g/l yeast extract, 5g/l NaCl supplemented with 8θml 0.25# haemin in 1/lOON NaOH. Strains of S. tvphimurium used were SL3261 and LB5010 as described by Hosieth & Stocker (1981) Nature (London) 291: p238-239 and Maskell et al (1987) Microb. Pathog. 2 pp211-221 respectively and these and E.coli JM109 were cultured and stored as described by Sambrook et al (1989) Molecular Cloning Manual. Cloning of cafl: DNA was isolated from Y. pestis by the method of Marmur et al (1961) J. Mol. Biol. 3 - PP 208-218. A DNA fragment encoding the open reading frame of cafl minus its signal sequence was amplified from this using the polymerase chain reaction (PCR) . Oligonucleotides were prepared with a Beckman 200A DNA synthesiser for use in the PCR. EXAMPLE 1: pFGAL2a construct: Oligonucleotide GATCGAGCTCGGCAGATTTAACTGCAAGCACC (SEQ ID No 1) was synthesised corresponding to the first 21 bases of cafl immediately following the nucleotides encoding the signal sequence with an additional 5' region encoding a Sacl site and the complimentary oligonucleotide CAGGTCGAGCTCGTCGACGGTTAGGCTCAAAGTAG (SEQ ID No 2) corresponding to the sequence which encodes a putative 'stem loop' structure downstream of the cafl termination codon with an added 5' region encoding Sacl and Accl sites. A DNA fragment was obtained after 35 cycles of amplification (95°C, 15 sees; 50°C, 15 sees; 72°C, 30 sees using a Perkin Elmer 96OO GeneAmp PCR system). The fragment was purified, digested with Sacl and Accl, ligated into a similarly digested pUClδ plasmid and transformed into E. coli JM109 by electroporation. Electroporation was carried out using a Biorad Gene Pulser with 0.2 cm cuvettes at 1.25kV, 25μF, δOOOhms with a time constant of 20. A pFGAL2a colony containing the cloned cafl gene was identified by PCR using an oligonucleotide TGGTACGCTTACTCTTGGCGGCTAT (SEQ ID No 3) corresponding to an internal region of the gene 128 to 153 nucleotides from the site identified as the signal sequence cleavage site (see Galyov et al (1990)) and the SEQ ID No 2. A culture of the E.coli - containing the pFGAL2a was grown at 37° c with shaking in Luria Broth containing lmM isopropyl-β-D-thiogalactopyranoside (IPTG) for 18 hours. Whole cell lysates and periplasmic and cytoplasmic fractions of the bacteria were prepared as described by Sambrook et al (1989). SDS-PAGE and Western blotting: SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting were performed as described by Hunter et al (1993) Infec. Immun. 61. 3958-3965- Blots were probed with polyclonal antisera raised in sheep (B283) against killed Y. pestis (EV76 strain grown at 37°C) and bound antibody was detected with a horseradish peroxidase-labelled donkey anti-sheep IgG (Sigma) . Expression of Fl in S. tvphimurium: The pFGAL2a plasmid was isolated using general techniques described in Sambrook et al (1989) Molecular Cloning; a Laboratory Manual. 2nd Edition. Cold Spring Harbour Laboratory, New York. Purified plasmid was electroporated into S. typhimurium LB5010 (restriction " , modification * ) and methylated pFGAL2a was subsequently isolated from the LB5010 for electroporation into S. typhimurium SL3261 (aro A " ). Periplasmic and cytoplasmic fractions were prepared for SDS-PAGE and Western blotting as described above. Stability of constructs: Five female Balb/c mice were inoculated intravenously with either 5xl0 5 or 5xl0 7 cfu S. tvphimurium containing pFGAL2a in 200μl phosphate buffered saline. Control mice were inoculated similarly with S. tvphimurium containing pUClδ with no insert. After 7 days the mice were killed by cervical dislocation and their livers and spleens removed. The organs were homogenised in 10ml phosphate buffered saline using a stomacher on maximum setting for 2 minutes and the homogenate was serially diluted in phosphate buffered saline and placed onto L agar or L agar containing 55ug ml "1 ampicillin. Challenf-fp gf -immunized mice: Male Balb/c mice were inoculated with three doses of S.tvphimurium containing pFGAL2a, intravenously (i.v.) with 5xl0 7 cfu bacteria at 7 day intervals, or intragastrically (i.g.) by intubation with 1 x IO 10 cfu on days 1, 7 and 21. Ampicillin was given subcutaneously for 5 days after each inoculation to stabilise the plasmid. Six weeks * after the last dose five mice from both the i.g. and intravenously inoculated groups were bled then killed by cervical dislocation and their livers and spleens taken. Livers were homogenised and plated onto L-agar to confirm that all Salmonella had been cleared. The remaining mice were challenged subcutaneously with 50LD- 0 of Y. pestis strain GB. Control mice were immunised i.v. and i.g. with S. tvphimurium containing pUClδ, intramuscularly with formaldehyde-killed plague vaccine (Cutter USP) or intraperitoneally with lOμg of purified Fl and survival rates assessed (see Table 1). EXAMPLE 2: pFSIG a construct: Construct plasmids and S. tvphimurium transformed by them were obtained using the method of Example 1 altered in so far as the PCR product was designed to be of SEQ ID No 2 after digestion with Sacl. The digested product was ligated into Sacl digested pFS2.2 giving the sequence SEQ ID No 5 located as shown in Figure I. This construct encodes for Fl which is expressed as a fusion with the E.coli LTB signal sequence. The primer used instead of SEQ ID No 1 was that of SEQ ID Nd 15. Example ' -*<: pFORFlb construct: Construct plasmids and S. tvphimurium transformed by them were obtained using the method of Example 1 altered in so far as the PCR primers were selected to produce an amplification product which after digestion with Sacl/Accl consisted of SEQ ID No 6. Primers used were of sequences SEQ ID NO 16 and SEQ ID No 17. This construct incorporates the cafl signal sequence in addition the mature protein sequence as shown in SEQ ID No 6 and Fig 1 which shows all bases downstream of the Sac I site used for digest. Protection provided by intravenous injection and oral delivery of S.tvphimurium transformed with plasmids containing various constructs of the invention and comparative protection afforded by direct administration of Fl and Cutter vaccines. S. typhimurium transformed as described above with either pFGAL2a, pFSIG3a, pFORFlb or unmodified pUClδ were administered to mice by intravenous or intragastric routes and compared in effect to intraperitoneal Fl and intramuscular Cutter vaccine as described above in Example 1. TABLE 1. CHALLENGE OF MICE WITH 50 x MICE LD 50 Y. PESTIS IV or IM*TREATMENT SURVIVORS ORAL TREATMENT SURVIVORS S.typhimurium 10/10 S.typhimurium 1/10 /pFSIG3a IV /pFSIG3a S.typhimurium 9/10 S.typhimurium 9/10 /pFGAL2a IV /pFGAL2a S.typhimurium 10/10 S.typhimurium 3/10 /pFORFlb IV /pFORFib S.typhimurium 0/10 S.typhimurium 0/5 /pUClδ IV /pUClδ Fl protein IP 6/8 Cutter vaccine 9/10 IM IV = intravenous IM = intramuscular IP = intraperitoneally EXAMPLE 4: Fl operon construct: Attempts to PCR replicate the entire Fl operon as one piece were unsuccessful, so a strategy was developed whereby it was amplified using PCR to produce two discrete fragments using primer pairs (A) of SEQ ID No 11 and 12 and (b) of SEQ No 13 and 14 respectively to produce fragments of 3-36kb and 1.89kb from Y. pestis MP6 template DNA. Maπnur extract of DNA was used without CsCl 2 purification. The PCR cycle conditions used were 96°C for 30 seconds, 57°C for 30 seconds and 72°C for 1 minute; total of 30 cycles. These two fragments were digested using Nhel and joined together. This fused fragment, encoding the full length operon (5-25kb), was digested with EcoRl and Sail and then cloned into a number of vectors. When this fragment was cloned into pBR322 and expressed in E. coli. S. typhimurium LB5010 or SL3261 instability of the recombinant plasmid was noted. To circumvent this problem the operon was cloned into plasmid pLG339. a low copy number plasmid km R . The entire Fl operon was also been inserted into AroC gene on the chromosome of S. typhimurium using vector pBRD10δ4. The positions of the primers, restriction sites and caflR to cafl are shown in Figure 2. Full protection (4/4) of mice challenged with with IO 4 lethal doses of Y. pestis was provided on administration of these transformed organisms. SEQUENCE LISTING 1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: THE SECRETARY OF STATE FOR DEFENCE IN HER BRITANNIC MAJESTY (B) STREET: WHITEHALL (C) CITY: LONDON (E) COUNTRY: UNITED KINGDOM (F) POSTAL CODE (ZIP) :. SW1A 2HB (A NAME: RICHARD WILLIAM TITBALL (B STREET: CBDE PORTON DOWN (C CITY: SALISBURY (D STATE: WILTSHIRE (E COUNTRY: UNITED KINGDOM (GB) (F POSTAL CODE (ZIP): SP4 OJQ (A NAME: ETHEL DIANE WILLIAMSON (B STREET: CBDE PORTON DOWN (C CITY: SALISBURY (D STATE: WILTSHIRE (E COUNTRY: UNITED KINGDOM (GB) (F POSTAL CODE (ZIP): SP4 OJQ (A NAME: SOPHIE EMMA CLARE LEARY (B STREET: CBDE PORTON DOWN (C CITY: SALISBURY (D STATE: WILTSHIRE (E COUNTRY: UNITED KINGDOM (GB) (F POSTAL CODE (ZIP) : SP4 OJQ (A NAME: PETRA CLAIRE FARQUHAR OYSTON (B STREET: CBDE PORTON DOWN (C CITY: SALISBURY (D STATE: WILTSHIRE (E COUNTRY: UNITED KINGDOM (GB) (F POSTAL CODE (ZIP): SP4 OJQ (A NAME: ANGELA HOWELLS (B STREET: CBDE PORTON DOWN (C CITY: SALISBURY (D STATE: WILTSHIRE (E COUNTRY: UNITED KINGDOM (GB) (F POSTAL CODE (ZIP): SP4 OJQ (ii) TITLE OF INVENTION: VACCINE COMPOSITIONS (iii)NUMBER OF SEQUENCES: 14 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release §1.0, Version §1.25 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: GB 9326425-7 (B) FILING DATE: 24-DEC-1993 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Yersinia pestis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GATCGAGCTC GGCAGATTTA ACTGCAAGCA CC 32 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Yersinia pestis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CAGGTCGAGC TCGTCGACGG TTAGGCTCAA AGTAG 3 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iii)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)ORGANISM: Yersinia pestis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TGGTACGCTT ACTCTTGGCG GCTAT 25 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 541 base pairs (B)TYPE: nucleic acid (C)STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iii)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)ORGANISM: Yersinia.pestis (ix) FEATURE: (A)NAME/KEY: CDS (B)LOCATION: 2..454 (ix) FEATURE: (A)NAME/KEY: misc recomb (B)LOCATION: 1..6 (ix) FEATURE: (A)NAME/KEY: misc recomb (B)LOCATION: 536..5-41 (xi) SEQUENCE : DESCRIPTION: SEQ ID NO: G AGC TCG GCA GAT TTA ACT GCA AGC ACC ACT GCA ACG GCA ACT CTT 46 Ser Ser Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu 1 . 5 10 15 GTT GAA CCA GCC CGC ATC ACT ATT ACA TAT AAG GAA GGC GCT CCA ATT 94 Val Glu Pro Ala Arg He Thr He Thr Tyr Lys Glu Gly Ala Pro He 20 25 30 ACA ATT ATG GAC AAT GGA AAC ATC GAT ACA GAA TTA CTT GTT GGT ACG 142 Thr He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr 35 40 45 CTT ACT CTT GGC GGC TAT AAA ACA GGA ACC ACT AGC ACA TCT GTT AAC 190 Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn 50 55 60 TTT ACA GAT GCC GCG GGT GAT CCC ATG TAC TTA ACA TTT ACT TCT CAG 238 Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin 65 70 75 GAT GGA AAT AAC CAC CAA TTC ACT ACA AAA GTG ATT GGC AAG GAT TCT 286 Asp Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys Asp Ser 80 85 90 95 AGA GAT TTT GAT ATC TCT CCT AAG GTA AAC GGT GAG AAC CTT GTG GGG 334 Arg Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly 100 105 110 GAT GAC GTC GTC TTG GCT ACG GGC AGC CAG GAT TTC TTT GTT CGC TCA 382 Asp Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val Arg Ser 115 120 125 ATT GGT TCC AAA GGC GGT AAA CTT GCA GCA GGT AAA TAC ACT GAT GCT 30 He Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala 130 135 140 GTA ACC GTA ACC GTA TCT AAC CAA TAATCCATAT AGATAATAGA TAAAGGAGGG 484 Val Thr Val Thr Val Ser Asn Gin 145 150 CTATTATGCC CTCCTTTAAT ATTTATGAAT TATCCTACTT TGAGCCTAAC CGTCGAC 4l (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 151 amino acids (B)TYPE: amino acid (D)TOPOLOGY: linear (ii)MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 5: Ser Ser Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val 1 5 10 15 Glu Pro Ala Arg He Thr He Thr Tyr Lys Glu Gly Ala Pro He Thr 20 25 30 He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr Leu 35 40 45 Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe 50 55 60 Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin Asp 65 70 75 80 Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys Asp Ser Arg 85 90 95 Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp 100 105 HO Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val Arg Ser He 115 120 125 Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val 130 135 140 Thr Val Thr Val Ser Asn Gin 145 150 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 542 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iii)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Yersinia pe (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3.-455 (ix) FEATURE: (A) NAME/KEY: misc recomb (B) LOCATION: 1..6 (ix) FEATURE: (A) NAME/KEY: misc recomb (B) LOCATION: 536.-541 (xi) SEQUENCE : DESCRIPTION: SEQ ID NO: 6: GA GCT CCC GCA GAT TTA ACT GCA AGC ACC ACT GCA ACG GCA ACT CTT 47 Ala Pro Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu 1 5 10 15 GTT GAA CCA GCC CGC ATC ACT ATT ACA TAT AAG GAA GGC GCT CCA ATT 5 Val Glu Pro Ala Arg He Thr He Thr Tyr Lys Glu Gly Ala Pro He 20 25 30 ACA ATT ATG GAC AAT GGA AAC ATC GAT ACA GAA TTA CTT GTT GGT ACG 143 Thr He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr 35 40 45 CTT ACT CTT GGC GGC TAT AAA ACA GGA ACC ACT AGC ACA TCT GTT AAC 191 Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn 50 55 60 TTT ACA GAT GCC GCG GGT GAT CCC ATG TAC TTA ACA TTT ACT TCT CAG 239 Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin 65 70 75 GAT GGA AAT AAC CAC CAA TTC ACT ACA AAA GTG ATT GGC AAG GAT TCT 287 Asp Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys Asp Ser 80 85 90 95 AGA GAT TTT GAT ATC TCT CCT AAG GTA AAC GGT GAG AAC CTT GTG GGG 335 Arg Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly 100 105 HO GAT GAC GTC GTC TTG GCT ACG GGC AGC CAG GAT TTC TTT GTT CGC TCA 383 Asp Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val Arg Ser 115 120 125 ATT GGT TCC AAA GGC GGT AAA CTT GCA GCA GGT AAA TAC ACT GAT GCT 431 He Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala 130 135 140 GTA ACC GTA ACC GTA TCT AAC CAA TAATCCATA TAGATAATAG ATAAAGGAGG 484 Val Thr Val Thr Val Ser Asn Gin 145 150 GCTATTATGC CCTCCTTTAA TATTTATGAA TTATCCTACT TTGAGCCTAA CCGTCGAC 542 (2) INFORMATION FOR SEQ ID NO: 7 - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 151 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: J : Ala Pro Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val 1 5 10 15 Glu Pro Ala Arg He Thr He Thr Tyr Lys Glu Gly Ala Pro He Thr 20 25 30 He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr Leu 35 40 45 Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe 50 55 60 Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin Asp 65 70 75 80 Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys Asp Ser Arg 85 90 95 Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp 100 105 110 Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val Arg Ser He 115 120 125 Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val 130 135 140 Thr Val Thr Val Ser Asn Glu 145 150 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 542 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Yersinia pestis (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 2..7 (ix) FEATURE: (A) NAME/KEY: misc_recomb (B) LOCATION 1..6 (ix) FEATURE: (A) NAME/KEY misc recomb (B) LOCATION 536..541 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION 21..530 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: G AGC TCG TAGGAGGTAA TAT ATG AAA AAA ATC AGT TCC GTT ATC GCC ATT 50 Ser Ser Met Lys Lys He Ser Ser Val He Ala He 1 1 5 10 GCA TTA TTT GGA ACT ATT GCA ACT GCT AAT GCG GCA GAT TTA ACT GCA 98 Ala Leu Phe Gly Thr He Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala 15 20 25 AGC ACC ACT GCA ACG GCA ACT CTT GTT GAA CCA GCC CGC ATC ACT CTT 146 Ser Thr Thr Ala Thr Ala Thr Leu Val Glu Pro Ala Arg He Thr Leu 30 35 40 ACA TAT AAG GAA GGC GCT CCA ATT ACA ATT ATG GAC AAT GGA AAC ATC 194 Thr Tyr Lys Glu Gly Ala Pro He Thr He Met Asp Asn Gly Asn He 45 50 55 GAT ACA GAA TTA CTT GTT GGT ACG CTT ACT CTT GGC GGC TAT AAA ACA 242 Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr 60 65 70 GGA ACC ACT AGC ACA TCT GTT AAC TTT ACA GAT GCC GCG GGT GAT CCC 290 Gly Thr Thr Ser Thr Ser Val Asn Phe Thr Asp Ala Ala Gly Asp Pro 75 80 65 90 ATG TAC TTA ACA TTT ACT TCT CAG GAT GGA AAT AAC CAC CAA TTC ACT 338 Met Tyr Leu Thr Phe Thr Ser Gin Asp Gly Asn Asn His Gin Phe Thr 95 100 105 ACA AAA GTG ATT GGC AAG GAT TCT AGA GAT TTT GAT ATC TCT CCT AAG 386 Thr Lys Val He Gly Lys Asp Ser Arg Asp Phe Asp He Ser Pro Lys 110 115 120 GTA AAC GGT GAG AAC CTT GTG GGG GAT GAC GTC GTC TTG GCT ACG GGC 434 Val Asn Gly Glu Asn Leu Val Gly Asp Asp Val Val Leu Ala Thr Gly 125 130 135 AGC CAG GAT TTC TTT GTT CGC TCA ATT GGT TCC AAA GGC GGT AAA CTT 482 Ser Gin Asp Phe Phe Val Arg Ser He Gly Ser Lys Gly Gly Lys Leu 140 145 150 GCA GCA GGT AAA TAC ACT GAT GCT GTA ACC GTA ACC GTA TCT AAC CAA 530 Ala Ala Gly Lys Tyr Thr Asp Ala Val Thr Val Thr Val Ser Asn Gin 155 1 0 165 170 TAATCCATAT AG 542 (2) INFORMATION FOR SEQ ID NO: 9 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 Ser Ser 1 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 170 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Met Lys Lys He Ser Ser Val He Ala He Ala Leu Phe Gly Thr He 1 10 15 Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala 20 25 30 Thr Leu Val Glu Pro Ala Arg He Thr Leu Thr Tyr Lys Glu Gly Ala 35 40 45 Pro He Thr He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val 50 55 60 Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser 65 70 75 80 Val Asn Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr 85 90 95 Ser Gin Asp Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys 100 105 HO Asp Ser Arg Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu 115 120 125 Val Gly Asp Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val 130 135 140 Arg Ser He Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr 145 150 155 160 Asp Ala Val Thr Val Thr Val Ser Asn Gin 165 170 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iϋ) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Yersinia pestis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TCGCCCGGGA ATTCCGAACA TAAATCGGTT CAGTGGCC 38 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iii)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Yersinia pestis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GGCGTATTCC TCGCTAGCAA TGTTTAACG 29 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Yersinia pestis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: ATCGTTAAAC ATTGCTAGCG AGGAATACGC C 31 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iii)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)ORGANISM: Yersinia pestis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GATAGATCTG TCGACTGAAC CTATTATATT GCTTCGCGC 39 (2) INFORMATION FOR SEQ ID NO: 15 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOPTHETICAL: NO (iii)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Yersinia Pestis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CTAGGAGCTC CCGCAGATTT AACTGCAAGC 30 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic). (iii)HYPOPTHETICAL: NO (iii)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Yersinia Pestis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GATCGAGCTC GTAGGAGGTA ATATATGAAA 30 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOPTHETICAL: NO (iii)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Yersinia Pestis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CAGGTCGAGC TCGTCGACCT ATATGGATTA TTGGT 35

Claims: CLAIMS . 1. Recombinant DNA that when incorporated into the DNA of a microorganism is capable of transforming that microorganism such that it is enabled to express a protein which produces a protective immune response against Yersinia pestis in a human or animal body when the microorganism is administered by oral route. 2. A plasmid capable of transforming a microorganism such that it is enabled to express a protein which produces a protective immune response against Yersinia pestis in a human or animal body when the microorganism is administered by oral route. 3. A microorganism comprising recombinant DNA, or a plasmid comprising recombinant DNA, whereby it is enabled to express a protein which produces a protective immune response against Yersinia pestis in a human or animal body when administered by oral route. 4. A microorganism as claimed in Claim 3 characterised in that it is a human or animal gut colonising microorganism. 5. Recombinant DNA, a plasmid or a microorganism as claimed in any one of claims 1 to 4 wherein the transformed microorganism maintains its ability to colonise the human or animal gut. 6. Recombinant DNA, a plasmid or a microorganism as claimed in Claim 1, 2, 3. 4 or 5 wherein the protein which produces the immune response comprises all or part of the Fl protein of Yersinia pestis. 7. Recombinant DNA comprising a DNA sequence as described in SEQ ID No 4. 8. Recombinant DNA as claimed in claim 1 comprising the Fl operon of Yersinia pestis including the caflR, caflM, caflA and cafl gene subunits. 9. Recombinant DNA as claimed in claim 8 wherein the Fl operon is derived by PCR amplification of Yersinia pestis template DNA using primer pairs of SEQ ID No 11 and 12 and SEQ ID No 13 and 14 to produce two discrete fragments, these fragments digested using Nhel and then joined to provide a single Fl operon containing fragment. 10. A plasmid comprising recombinant DNA as claimed in claim 8 or 9- 11. A plasmid as claimed in claim 10 being a low copy number plasmid. 12. A plasmid as claimed in claim 10 or 11 wherein the plasmid is PLG339 or pBRD1084. 13. A plasmid as claimed in Claim 2 comprising a DNA sequence as described in SEQ ID No 4. 14. A plasmid as claimed in Claim 2 or Claim 13 characterised in that it comprises a lacZ promoter upstream of a sequence encoding for all or part of the Fl antigen. 15. A plasmid as claimed in Claim 14 characterised in that it comprises a pUClδ vector that has all or part of the cafl sequence inserted downstream of the lacZ promoter. 16. A microorganism as claimed in Claim 3 or 4 containing recombinant DNA comprising a DNA sequence as described in SEQ ID No 4 or as claimed ih claim δ or 9- 17. A microorganism as claimed in Claim 3 or 4 containing a plasmid as claimed in any one of claims 7 to 15. 18. A microorganism as claimed in Claim 3. 4, 16 or 17 being an attenuated microorganism not capable of causing disease in humans or animals. 19. A microorganism as claimed in Claim 3. 4, 10, 11 or 12 being an aro A and/or aro C mutant. 20. A microorganism as claimed in Claim 18 or 19 being a Salmonella. 21. A microorganism as claimed in Claim 20 being a Salmonella typhimurium or a Salmonella typhi. 22. A microorganism as claimed in any one of claims 3« 4, lδ, 19, 20 or 21 wherein a recombinant DNA of claim 8 or 9 is integrated directly into the microorganisms chromosomal DNA. 23. A vaccine comprising a microorganism as claimed in any one of Claim 3. 4 or 16 to 22 together with a pharmaceutically acceptable carrier. 24. A recombinant DNA, plasmid, microorganism or vaccine according to any one of claims 1 to 23 as described in Example 1 or Example 4.

Inventors: HOWELLS ANGELA, LEARY SOPHIE EMMA CLARE, OYSTON PETRA CLAIRE FARQUHAR, TITBALL RICHARD WILLIAM, WILLIAMSON ETHEL DIANE, HOWELLS, ANGELA, LEARY, SOPHIE, EMMA, CLARE, OYSTON, PETRA, CLAIRE, FARQUHAR, TITBALL, RICHARD, WILLIAM, WILLIAMSON, ETHEL, DIANE
Applicants: HOWELLS ANGELA, LEARY SOPHIE EMMA CLARE, OYSTON PETRA CLAIRE FARQUHAR, SECR DEFENCE BRIT, TITBALL RICHARD WILLIAM, WILLIAMSON ETHEL DIANE, HOWELLS, ANGELA, LEARY, SOPHIE, EMMA, CLARE, OYSTON, PETRA, CLAIRE, FARQUHAR, THE SECRETARY OF STATE FOR DEFENCE IN HER BRITANNI, TITBALL, RICHARD, WILLIAM, WILLIAMSON, ETHEL, DIANE
Application year: 1994
Main classification: C12N15
IPC codes: A61K39, A61P31, C07K14, C12N1, C12N15